Method to select animals with a high capacity of embryo production

ABSTRACT

The present invention provides a method to select a non-human female animal subject having the capacity to produce appropriate number of transferable embryos or fertilizable oocytes in said animal, said method comprising the determination of the anti-Mullerian hormone concentration in a biological sample before an ovarian superovulatory treatment. The present invention is further directed to the use of AMH as a predictive marker of number of transferable embryos or fertilizable oocytes able to be produced in a non-human female animal.

The invention relates to a method to select a non-human female animalsubject having the capacity to produce appropriate number of embryos orfertilizable oocytes.

In cattle, Multiple Ovulation and Embryo Transfer (MOET) programs havebecome a large international business. Embryo transfer has become auseful tool to accelerate the genetic progress and a national andinternational diffusion thereof. The technology is well established andparticipates to genetic selection strategies and crossbreeding schemesto improve zootechnical characteristics of dairy and beef breeds byenabling to amplify the lineage of chosen females. More than 500,000embryos are produced annually from superovulated (FSH-stimulated) cowsworldwide. Currently, more than 90% of future bull reproducers subjectedto testing are produced by MOET technology. However, the number oftransferable embryos (around 6 per superovulated donor cow) has notchanged markedly in the last twenty years and the use of MOET technologyin the animal industries is approaching a plateau. In fact, about 20% ofcows don't produce any embryo after superovulatory treatments and embryoproduction rate is also very variable between individuals and difficultto predict. The absence of prevision of the capacity of a female animalto produce embryos induces supplementary cost in the management ofselection schemes (unnecessary mobilization of technicians for animalsproducing low numbers of transferable embryos) and reduces the possibleuse of related techniques to embryo transfer such as embryo sexing.

Currently, the count of antral follicles by ovarian ultrasonography isthe only method to select foreword “good donors of embryos” in a cattleherd (Durocher et al., 2006, Theriogenology 65: 102-115; Kawamata, 1994,J Vet Med Sci 56: 965-967). This method consists in transrectalultrasonography. It needs the simultaneous presence of 2 competentexperimenters for acquisition of ovarian images and requires specialequipments for acquisition and treatment of video images. Moreover, thenumber of antral follicles in ovaries is subjected to cyclic variationswith time (2 to 4 follicular waves can be observed during a 21-daysexual cycle in cow) that makes it more difficult to interpret obtainedresults. Therefore, since a long time, skilled person is searching foran easy and effective method to select the female animals that canproduce appropriate number of embryos or fertilizable oocytes.

Recently, attention has been focused on the anti-Mullerian hormone (AMH)in the context of treatment of anovulatory infertility in woman.Numerous clinical studies have shown that AMH is the best endocrinemarker of the ovarian follicular reserve in human. WO 03/016514 reporteda method for predicting and monitoring a woman's response to fertilitytreatments, based on the measurement of AMH level in said woman.

AMH is a glycoprotein of 140 kDa belonging to the transforming growthfactor β family (TGFβ), that is expressed only in the gonads. It wasoriginally identified in connection with its role in male fetal sexdifferentiation during embryonic development, but later studies haveshown that AMH exerts inhibitory effects on the development and functionof reproductive organs in both sexes. In the ovary, AMH expression isrestricted to a single cell type, i.e. granulosa cells (Vigier et al.,1984, Endocrinology 114: 1315-1320; Takahashi et al., 1986, Biol Reprod35: 447-453). AMH expression is the highest in granulosa cells ofpreantral and small antral follicles, decreases during terminalfollicular growth, and low AMH concentrations have been found infollicular fluid of large antral and preovulatory follicles (Monniaux etal., 2008, Biol Reprod 79: 387-396). This pattern of expression ingranulosa cells of growing follicles makes AMH an ideal marker for thesize of the ovarian follicle pool. The inventors have previouslyobserved that in cow, as in human, plasma concentration of AMH is a goodmarker of the status of the follicular population able to respond to FSH(follicle-stimulating hormone) treatment and to ovulate (Rico et al.,2009, Biol Reprod 80: 50-59).

Ovulation is a first step that is necessary but not sufficient to obtainfertilizable oocytes and transferable embryos. The other steps,concerning survival of the ovulated oocyte in the oviduct, transit andsurvival of sperm in the female genital tract after insemination,fertilization and early embryo development up to the morula or theblastocyst stage, are also decisive for production of good qualityoocytes and embryos. Previous results have shown that superovulation candecrease developmental competence of bovine oocytes (Lonergan et al.,1994, Mol Reprod Dev 37: 48-53; Blondin et al., 1996, Theriogenology 46:1191-1203) and that a high ovulation capacity can sometime produce a lowproportion of transferable embryos (Ireland et al., 2007, Human Reprod22: 1687-1695). These results might be explained by a poorsynchronization between ovulation and insemination timing, or byabnormal endocrine profiles of steroids that can affect both spermtransport and embryo development and survival in the case of multipleovulations (Greve and Callesen, Reprod Nutr Dev 41: 451-459). Due to themultiplicity of hormones, growth factors and cell-cell interactionsinfluencing the number and the quality of oocytes and embryos, up tonow, there is no reliable method able to predict the number offertilizable oocytes and transferable embryos that can be produced by apotential donor cow after superovulatory treatment.

Now, the inventors found out that AMH concentration in plasma could benot only an endocrine marker of follicular population, but also afaithful predictive marker of the numbers of transferable embryos orfertilizable oocytes able to be produced by an individual animal aftersuperovulatory treatment. The present invention proposes, for the firsttime, a prognosis method to determine the capacity of embryo or oocyteproduction by potential embryo or oocyte donor animals, from the resultof measurement of AMH concentration in a biological sample of thesepotential donors. The inventors showed that plasma or serum AMHconcentration can stay stable in individual animals over several months,and this property makes this prognosis to be performed by only one bloodtest per animal.

In the first aspect, the present invention provides a method to select anon-human female animal subject having the capacity to produceappropriate number of transferable embryos or fertilizable oocytes insaid animal, said method comprising the determination of theanti-Mullerian hormone concentration in a biological sample before anovarian superovulatory treatment.

In the context of the present invention, “appropriate number oftransferable embryos or fertilizable oocytes” should be understood as atleast 10 transferable embryos, or at least 10 fertilizable oocytes.

The transferable embryos are those qualified as “good” embryos,according to the classic morphological standard of shape and color usedin field work (Callesen et al., 1995, J Anim Sci 73: 1539-1543) and thedefinitions developed by the International Embryo Transfer Society.According to the invention, production of embryos or oocytes is achievedaccording to techniques well-known in prior art. An embryo donor animalwhich can produce more than 10 transferable embryos after asuperovulatory treatment is defined as a good embryo donor. An embryodonor which produces less than 5 transferable embryos is defined as apoor embryo donor.

The fertilizable oocytes are the oocytes collected from the follicles ofa female animal, which are capable to be fertilized in vitro and to giverise to transferable embryos.

The embryos can be produced by in vivo fertilization through a naturalor artificial insemination of a female animal which has formerlyreceived an ovarian superovulatory treatment. Embryos produced in thisway are collected through genital tract. The embryos can be alsoproduced by in vitro fertilization of oocytes collected by follicularpuncture (Ovum Pick-Up technique, or OPU) from the ovaries of a femaleanimal which has formerly received an ovarian superovulatory treatment.Oocytes (immature ova) are collected from the follicles in the ovariesby aspiration using ultrasonic guidance through the vaginal wall. Theoocytes are matured in the laboratory for 24 hours then fertilized andembryos are cultured for a further seven-day period before beingtransferred to prepared recipients or frozen for use at a later date.

In a particular embodiment, the method according to the presentinvention comprises the following steps:

-   -   a) sampling of a biological sample,    -   b) measuring in said biological sample the anti-Mullerian        hormone concentration,    -   c) comparing said concentration with two, respectively “high”        and “low”, reference values        and wherein if the anti-Mullerian hormone concentration is        greater than the “high” reference value, then the animal is        considered as having a good capacity to produce transferable        embryos or fertilizable oocytes; conversely, if the        anti-Mullerian hormone concentration is lower than the “low”        reference value, then the animal is considered as having a poor        capacity to produce transferable embryos or fertilizable        oocytes.

The said biological sample can be any biological fluids selected fromthe group comprising plasma, serum, milk, urine or saliva.

In a preferred embodiment according to the invention, the saidbiological sample is plasma or serum.

According to the invention, the AMH concentrations can be measured byany classical dosage method disclosed in literature or known by theskilled person.

In another preferred embodiment of the present invention, the animal isa ruminant.

In a more preferred embodiment of the present method according to theinvention, the ruminant is selected from the group comprising bovine andcaprine species.

The term “reference value” is used in the present invention to define athreshold concentration of AMH over (for the “high” reference) or under(for the “low” reference) which an appropriate or unappropriate numberof transferable embryos or fertilizable oocytes is respectivelypredicted to be recovered from a donor animal with a confidence levelstatistically higher than 95%. These reference values have beendetermined from statistic analysis of a number of measurements of bothAMH concentrations and numbers of embryos produced in differentsubjects.

According to the present invention, the “high” reference value ofanti-Mullerian hormone concentration in plasma of bovine species iscomprised between 160 pg/ml to 200 pg/ml, preferably 180 pg/ml forproduction of more than 10 transferable embryos or fertilizable oocytesper ovarian superovulatory treatment, and the “low” reference value ofanti-Mullerian hormone concentration in plasma of bovine species iscomprised between 80 pg/ml to 110 pg/ml, preferably 90 pg/ml forproduction of less than 5 transferable embryos or fertilizable oocytesper ovarian superovulatory treatment.

In the second aspect, the present invention is also directed to the useof anti-Mullerian hormone as a predictive marker of the number oftransferable embryos or fertilizable oocytes able to be produced in anon-human female animal subject.

The invention is illustrated by the following examples and FIGS. 1 to 5.

FIG. 1A illustrates the relationship between the average number ofembryos produced per female and their AMH concentration in plasma(r=0.49, p<0.001). Blood sample for each 45 cows was taken during theirfirst lactation and then these cows have been submitted to ovariansuperovulatory treatment, artificial insemination and embryo collect.

FIG. 1B illustrates the relationship between the maximal number ofembryos produced per female and their AMH concentration in plasma(r=0.58, p<0.0001). Blood sample for each 45 cows was taken during theirfirst lactation and then these cows have been submitted to ovariansuperovulatory treatment, artificial insemination and embryo collect.

FIG. 1C illustrates the relationship between AMH concentration in plasmaand the average number of embryos produced per female. 45 cows weredivided into three groups according to their AMH concentration, group[0-100] (n=16), group [100-200] (n=15) and group [>200] (n=14). *p<0.05,**p<0.01 vs. group [0-100].

FIG. 1D illustrates the relationship between AMH concentration in plasmaand the maximal number of embryos produced per female. 45 cows weredivided into three groups according to their AMH concentration inplasma, as defined in legend of FIG. 1C. *p<0.05, **p<0.01 vs. group[0-100].

FIG. 2A illustrates the relationship between the average number oftransferable embryos produced per female and their AMH concentration inplasma (r=0.32, p<0.05). Blood sample for each 45 cows was taken duringtheir first lactation and then these cows have been submitted to ovariansuperovulatory treatment, artificial insemination and embryo collect.

FIG. 2B illustrates the relationship between the maximal number oftransferable embryos produced per female and their AMH concentration inplasma (r=0.38, p=0.01). Blood sample for each 45 cows was taken duringtheir first lactation and then these cows have been submitted to ovariansuperovulatory treatment, artificial insemination and embryo collect.

FIG. 2C illustrates the relationship between AMH concentration in plasmaand the average number of transferable embryos produced per female. 45cows were divided into three groups according to their AMHconcentration, as defined in legend of FIG. 1C. *p<0.05 vs. group[0-100].

FIG. 2D illustrates the relationship between AMH concentration in plasmaand the maximal number of transferable embryos produced per female. 45cows were divided into three groups according to their AMHconcentration, as defined in legend of FIG. 1C. *p<0.05, vs. group[0-100].

FIG. 3A illustrates mean embryo production (total embryos andtransferable embryos) per female. 45 cows were divided in two groupsaccording to their capacity (C) of embryo production, group [C<10](n=13) and group [C>10] (n=32). Capacity was defined as the maximalnumber of transferable embryos that could be collected per female aftersuperovulatory treatment. ***p<0.001 vs. group [C<10].

FIG. 3B illustrates mean AMH concentration in plasma. 45 cows weredivided in two groups according to their capacity (C) of embryoproduction, as defined in legend of FIG. 3A. **p<0.01 vs. group [C<10].

FIG. 4A illustrates individual variations in AMH concentrations of 5cows with low mean AMH concentrations (<150 pg/ml), measured before eachrepetition of ovarian superovulatory treatment and OPU (Ovum Pick-Up).

FIG. 4B illustrates individual variations in AMH concentrations of 5cows with high mean AMH concentrations (>150 pg/ml), measured beforeeach repetition of ovarian superovulatory treatment and OPU.

FIG. 5A illustrates the effect of OPU repetitions on AMH concentrationsbefore treatment (black circles, right scale) and on the number of largefollicles at OPU (black bars, left scale). Cows were treated repeatedly(n=4 to 11 repetitions per cow). The horizontal axis represents the OPUrepetition number. The number of studied cows is indicated betweenbrackets.

FIG. 5B illustrates the value of the coefficient of repeatability of AMHconcentration before treatment (black circles) and of the number oflarge follicles at OPU (empty squares). Cows were treated repeatedly(n=4 to 11 repetitions per cow). The horizontal axis represents thenumber of OPU that were carried out per cow. The number of studied cowsis indicated between brackets.

EXAMPLE 1 AMH Concentration in Plasma and Production of Embryos AfterSuperovulation in Prim'Holstein×Normande Cows

Materials and Methods

Animals and Experimental Design

Forty-five crossbred Prim'Holstein×Normande dairy cows, 2 to 4 yearsold, were submitted to a blood test during their first lactation,between 60 and 90 days after calving, to determine their AMHconcentration in plasma. Blood (2 to 4 ml) was collected in anheparinized tube, then plasma was recovered after centrifugation andstored at −20° C. until AMH assay.

Afterwards, these cows were submitted to repeated ovarian superovulatorytreatments (1 to 9 repetitions of treatments per animal, mean number ofrepetitions per animal=5.4), each followed by artificial inseminationand embryo collect. Each superovulatory treatment consisted inadministration of 32 mg of FSH (follicle-stimulating hormone,STIMOFUL®), given as twice daily injections over 4 days on a standarddecreasing dose schedule (Mapletoft et al., 2002, Reprod Nutr Dev 42:601-611). The interval between blood recovery during lactation and thefirst embryo collect varied in the range of [3-30] months between cows(mean interval=17.2 months). Embryos were collected by flushing uterinehorns, counted and their quality was evaluated according to classicmorphological criteria (Callesen et al., 1995, J Anim Sci 73:1539-1543). Embryos with quality 1 to 3 according to the criteria ofCallesen et al. (1995) were defined as transferable (i.e. good) embryos.

AMH Assay

AMH was measured with the Active MIS/AMH ELISA kit (Beckman CoulterFrance, Roissy CDG, France) as previously described (Monniaux et al.,2008, Biol Reprod 79: 387-396). AMH was measured on 50 μl of undilutedplasma. AMH concentrations were higher than the limit of detection ofthe assay (1 pg per well, corresponding to 0.020 ng/ml in plasmasamples) in all the plasmas tested. Intra-assay coefficient of variationwas found to be 11.8% and 3.6% for plasma samples containing 0.033 ng/mland 0.125 ng/ml AMH concentration, respectively.

Results

A close positive correlation was observed between AMH concentrationmeasured in plasma of cows during their first lactation and the averageand maximal number of embryos collected per animal (r=0.49, p<0.001 andr=0.58, p<0.0001, respectively, FIGS. 1A and 1B). The allocation ofanimals to 3 groups according to their AMH concentration discloses thatthe animals having AMH concentration between 100 to 200 pg/ml and higherthan 200 pg/ml have produced higher numbers of embryos than the animalshaving less than 100 pg/ml of AMH (group [100-200] vs. group [0-100],p<0.05; group [>200] vs. group [0-100], p<0.01 for both average andmaximal number of embryos, FIGS. 1C and 1D).

The average and maximal numbers of transferable embryos collected peranimal were also correlated to AMH concentration measured in plasma ofcows during their first lactation (r=0.32, p<0.05 and r=0.38, p=0.01,respectively, FIGS. 2A and 2B). The allocation of animals to 3 groupsaccording to their AMH concentration shows that the animals having AMHconcentration between 100 to 200 pg/ml and higher than 200 pg/ml haveproduced higher numbers of transferable embryos than the animals havingless than 100 pg/ml of AMH (group [100-200] vs. group [0-100], p<0.05;group [>200] vs. group [0-100], p<0.05 for both average and maximalnumber of transferable embryos, FIGS. 2C and 2D).

In a further analysis, animals were allocated to 2 groups according totheir capacity of production of transferable embryos. This capacity (C)was defined as the maximal number of transferable embryos that could becollected per animal after superovulatory treatment. Animals with a highcapacity of production of transferable embryos (good embryo donors,C>10) had higher AMH concentrations measured in plasma during theirfirst lactation (p<0.01, FIG. 3B), and they produced also a higheraverage number of total embryos (p<0.001) and transferable embryos(p<0.001, FIG. 3A) than embryo donors with C<10. Moreover, the goodembryo donors were able to produce in average 5 times more transferableembryos compared with poor embryo donors (donors with C<5) and had 2.7times higher AMH concentrations than the poor embryo donors in plasmaduring their first lactation.

In subsequent analyses, data modelling was carried out using differentlinear and non-linear models and best fitting was found using polynomialregression models. With these models and the present data, the choice ofa low reference value of AMH concentrations comprised between 80 and 110pg/ml allowed discarding 70% of poor embryo donors among the populationof poor embryo donors, and the choice of a high reference value of AMHconcentrations comprised between 160 and 200 pg/ml allowed selecting 60%of good embryo donors among the population of good embryo donors.

EXAMPLE 2 Repeatability of AMH Concentration in Plasma and FollicularResponse in Prim'Holstein Cows After Superovulation and OPU

Materials and Methods

Animals and Experimental Design

Thirteen Prim'Holstein cows were submitted to repeated ovariansuperovulatory treatments, each followed by OPU on the large follicles(diameter>6 mm) that were detected by ovarian ultrasonography. Eachsuperovulatory treatment consisted in administration of 32 mg of FSH(STIMOFUL®), given as twice daily injections over 4 days on a decreasingdose schedule and follicles were punctioned 48 h after the last FSHinjection. At each repetition of treatment, blood (2 to 4 ml) wascollected in an heparinized tube just before the first FSH injection(before treatment) and at the day of OPU (at OPU), then plasma wasrecovered after centrifugation and stored at −20° C. until AMH assay.

Cows entered the experimental protocol in January (6 cows), March (4cows) or May (3 cows). Then animals were treated and submitted to OPUrepeatedly (between 4 and 11 repetitions per cow) until December. Notreatment and OPU was carried out on cows during the hot season (Julyand August). Five cows with low follicular response (<15 large folliclesat each punction) left the protocol within 3 months only after entry.

AMH Assay

AMH concentration was measured in plasma with the Active MIS/AMH ELISAkit (Beckman Coulter France, Roissy CDG, France), as previouslydescribed in example 1.

Data Analysis

For group comparisons, data were analyzed using t-test or one-way ANOVA.In order to evaluate a seasonal effect, 5 periods of time were compared:January-February, March-April, May-June, September-October andNovember-December. The effects of repetition of treatments on the numberof large follicles and AMH concentrations were analyzed with repeatedmeasures ANOVA. The repeatability of each parameter was calculated asthe ratio of the between-animal variance to the sum of thebetween-animal and the residual variances. For correlation studies, thesignificance of the value of the correlation coefficient was consideredaccording to the Bravais-Pearson-r critical values. For all analyses,differences with p>0.05 were considered as not significant.

Results

A total number of 90 treatments, each followed by OPU, were carried outon the 13 cows. AMH concentrations increased following treatment(176.6±11.8 vs. 253.7±19.8 pg/ml, AMH before treatment vs. AMH at OPU,p<0.01) and a highly significant correlation was observed between AMHconcentrations measured before treatment and at the time of OPU (r=0.88,p<0.0001). The number of large follicles at OPU was significantlycorrelated with AMH concentrations before treatment (r=0.56, p<0.0001)and at the time of OPU (0.65, p<0.0001).

No seasonal effect was observed for both the number of large follicleson ovaries at OPU and AMH concentrations. For all parameters, there wasa tendency to an increase through time, likely related to the early exitof 5 low responding animals from the experimental protocol as saidabove, but this tendency was not significant (Table 1).

AMH concentrations before treatment showed low intra-animal variationswith time (FIGS. 4A and 4B). FIG. 5A and FIG. 5B illustrate individualrepeatability of AMH concentrations before treatment and folliclenumbers at OPU during the repeated sessions of OPU. There was nosignificant effect of the OPU repetition number on the number of largefollicles on ovaries at OPU and AMH concentrations before treatment(FIG. 5A). When data were analyzed for 4 (n=13 cows), 5 (n=10 cows), 6(n=9 cows), 7 (n=6 cows), 8 (n=5 cows) or 11 repeated OPU (n=3 cows),highly significant repeatability was observed in all analyses for AMHconcentrations before treatment (p<0.0001) (FIG. 5B). Similar resultswere observed for AMH concentrations at the time of OPU (data notshown).

TABLE 1 Effect of season on AMH concentrations before superovulatorytreatment and at OPU and on the number of large follicles at OPU Periodof time J-F M-A M-J J-A S-O N-D Number of cows 6 8 7 0 8 8 AMH beforetreatment 126.1 ± 28.5 136.6 ± 42.4 182.5 ± 29.6 NA 229.0 ± 52.3  256.4± 44.3 (pg/ml) AMH at OPU 164.3 ± 34.1 176.4 ± 59.7 257.2 ± 46.8 NA417.5 ± 101.8 340.5 ± 63.8 (pg/ml) Number of large 11.75 ± 226 11.65 ±2.03 14.19 ± 2.52 NA 12.19 ± 2.76  16.00 ± 3.70 follicles at OPU

1. A method for predicting the number of transferable embryos orfertilizable oocytes able to be produced by an individual non-humanfemale animal subject after superovulatory treatment, said methodcomprising the determination of the anti-Mullerian hormone concentrationin a blood sample of the subject before administration of an ovariansuperovulatory treatment.
 2. The method according to claim 1 whichcomprises: a) measuring the anti-Mullerian hormone concentration in theblood sample of the subject, and b) comparing said concentration with areference value, and wherein if the measured anti-Mullerian hormoneconcentration is greater than the reference value, then the subject isconsidered as having a good capacity to produce transferable embryos orfertilizable oocytes.
 3. The method according to claim 1: a) measuringthe anti-Mullerian hormone concentration in the blood sample of thesubject, and b) comparing said concentration with two, respectively“high” and “low”, reference values and wherein if the anti-Mullerianhormone concentration is greater than the “high” reference value, thenthe animal is considered as having a good capacity to producetransferable embryos or fertilizable oocytes; conversely, if theanti-Mullerian hormone concentration is lower than the “low” referencevalue, then the animal is considered as having a poor capacity toproduce transferable embryos or fertilizable oocytes.
 4. The method ofclaim 3 wherein the animal is a ruminant.
 5. The method of claim 4wherein the ruminant is of the bovine or caprine species
 6. The methodaccording to claim 1 wherein the anti-Mullerian hormone concentrationwhich is measured is the plasma or serum concentration of anti-Mullerianhormone.
 7. The method according to claim 1 wherein the animal is aruminant.
 8. The method according to claim 7 wherein the ruminant isselected from the group comprising bovine and caprine species.
 9. Themethod of claim 31 wherein the anti-Mullerian hormone concentration ismeasured by employing an ELISA assay.
 10. A method for improving thenumber of transferable embryos produced in non-human female animalswhich comprises the steps of: a) determining an anti-Mullerian hormoneconcentration in a blood sample of each of the non-human female animalsbefore administering an ovarian superovulatory treatment the non-humananimal by measuring the anti-Mullerian hormone concentration in theblood sample, b) comparing said concentration with a reference value,wherein if the measured anti-Mullerian hormone concentration is greaterthan the reference value, then the non-human animal is considered ashaving a good capacity to produce transferable embryos or fertilizableoocytes; c) administering superovulatory treatment to only thosenon-human female animals wherein the anti-Mullerian hormoneconcentration is greater than the reference value of anti-Mullerianhormone concentration indicating the animal has a good capacity toproduce transferable embryos or fertilizable oocytes; d) artificiallyinseminating the non-human female animals to which superovulatorytreatment was administered; and e) obtaining transferable embryos. 11.The method of claim 10 further comprising the step of obtaining a bloodsample from the non-human female animal subjects for determination ofthe anti-Mullerian hormone concentration in the blood sample.
 12. Themethod of claim 11 wherein the blood sample is plasma or serum.
 13. Themethod of claim 10 wherein comparing said concentration with a referencevalue comprises comparing said concentration with two, respectively“high” and “low,” reference values and wherein if the anti-Mullerianhormone concentration is greater than the “high” reference value, thenthe animal is considered as having a good capacity to producetransferable embryos or fertilizable oocytes; conversely, if theanti-Mullerian hormone concentration is lower than the “low” referencevalue, then the animal is considered as having a poor capacity toproduce transferable embryos or fertilizable oocytes.
 14. The method ofclaim 10 wherein the anti-Mullerian hormone concentration is measured byemploying an ELISA assay.
 15. A method for improving the number offertilizable oocytes produced in non-human female animals whichcomprises the steps of: a) determining an anti-Mullerian hormoneconcentration in a blood sample of each of the non-human female animalsbefore administering an ovarian superovulatory treatment to the animalsby measuring the anti-Mullerian hormone concentration in the bloodsample, b) comparing said concentration with a reference value, whereinif the measured anti-Mullerian hormone concentration is greater than thereference value, then the animal is considered as having a good capacityto produce transferable embryos or fertilizable oocytes; c)administering superovulatory treatment to only those non-human femaleanimals wherein the anti-Mullerian hormone concentration is greater thanthe reference value of anti-Mullerian hormone concentration indicatingthe animal has a good capacity to produce transferable embryos orfertilizable oocytes; and d) recovering fertilizable oocytes from theovaries of the non-human female animals to which superovulatorytreatment was administered.
 17. The method of claim 16 wherein comparingsaid concentration with a reference value comprises comparing saidconcentration with two, respectively “high” and “low,” reference valuesand wherein if the anti-Mullerian hormone concentration is greater thanthe “high” reference value, then the animal is considered as having agood capacity to produce transferable embryos or fertilizable oocytes;conversely, if the anti-Mullerian hormone concentration is lower thanthe “low” reference value, then the animal is considered as having apoor capacity to produce transferable embryos or fertilizable oocytes.18. The method of claim 16 wherein the blood sample is serum or plasma.19. The method of claim 16 wherein the anti-Mullerian hormoneconcentration is measured by employing an ELISA assay.
 20. The method ofclaim 16 further comprising the step of subjecting recoveredfertilizable oocytes to in vitro fertilization.